In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. This past year the BEMB has identified the cause of recessive OI, which has been sought since 1979. [unreadable] [unreadable] Structural defects of the heterotrimeric type I collagen molecule are well known to cause the dominant bone disorder osteogenesis imperfecta. A severe recessive form of OI was first postulated in 1979. More recently, investigators have noted that some patients with clinical OI do not have defects detected in the type I collagen genes during sequencing. These patients without mutations in collagen can be divided into those who have abnormal collagen biochemistry and those with normal electrophoretic migration of the collagen chains. We hypothesized that the cause of recessive OI with abnormal collagen biochemistry and normal collagen gene sequence would involve a gene(s) whose products interacted with type I collagen.[unreadable] [unreadable] Post-translational modifications of collagen are required for efficient folding, secretion and fibril assembly, including 4-hydroxylation of proline residues, hydroxylation of lysine residues and subsequent glycosylation of some hydroxylysines. Collagen is also modified by 3-hydroxylation of a single residue in the alpha 1(I) chain at Pro986, although the function of this specific modification has yet to be elucidated. Morello and colleagues (Morello, et al. Cell. 127, 291-304 (2006)) have generated a knock-out mouse for Cartilage-Associated Protein (CRTAP) with recessive bone dysplasia, characterized by defective osteoid formation and severe osteoporosis. CRTAP was also shown by the Bachinger lab to form a complex in the endoplasmic reticulum with cyclophilin B and prolyl 3-hydroxylase 1 (P3H1)/leprecan (encoded by LEPRE1) which interacts with and 3-hydroxylates collagen. These discoveries made CRTAP a suitable candidate for recessive OI.[unreadable] [unreadable] The BEMB cell repository contained cells from 10 patients who have overmodified collagen biochemistry, yet lack a mutation in either of the type I collagen genes. Of these 10 cases, we have identified three patients with null mutations for CRTAP, designated as type VII OI. Their clinical finding overlap lethal type II OI but with distinctive features, including white sclerae. Patients were screened for null mutations by real-time RT-PCR using primary fibroblast mRNA. Sequencing of the exons and surrounding intronic regions of the CRTAP gene identified homozygous or compound heterozygous mutations in patient genomic DNA, resulting in altered splicing and introduction of premature termination codons in transcripts. All three of the CRTAP null patients have a severe recessive form of OI, which is lethal in the first year of life. Their long bones are extremely osteoporotic and deformed, with prenatal fractures and a tubular shape, due to abnormal modelling. Additionally, they have a narrow thorax with multiple prenatal rib fractures. The mutations identified in CRTAP include a homozygous IVS1+1G>C splicing mutation, a homozygous Gln276Stop in exon 4 and a compound heterozygote of a 16 nt duplication in exon 1 in one allele, and a Met1Ile in the other allele. Parents of CRTAP probands were shown to be heterozygous carriers. Probands had absence of CRTAP protein on Western blot and absence of Pro986 hydroxylation on mass spectrometry analysis. [unreadable] [unreadable] We identified null LEPRE1 mutations in the remaining 7 cases, which we classified as type VIII OI. These probands have bone dysplasia overlapping lethal type II OI and severe type III OI, but with distinctive features, including white sclerae, as found in CRTAP defects. Patients were screened for null mutations by real-time RT-PCR using primary fibroblast mRNA. Sequencing of the exons and surrounding intronic regions of the LEPRE1 gene identified homozygous or compound heterozygous mutations in patient genomic DNA, resulting in altered splicing and introduction of premature termination codons in transcripts. The seven null LEPRE1 patients have severe to lethal recessive bone dysplasia, characterized by shortened long bones, generalized bone disorganization and have extremely low BMD (L1-L4 DEXA z-score = -7). Parents of LEPRE1 probands were shown to be heterozygous carriers. Furthermore, P3H1 protein was demonstrated to be absent in our patient fibroblasts on Western blots. Patient type I collagen has severely reduced or completely abolished 3-hydroxylation of the alpha 1(I) Pro986 residue, by mass spectrometry of collagen tryptic peptides. In contrast, the extent of overhydroxylation of proband collagens by lysyl hydroxylase is comparable to collagens containing a structural defect in the carboxyl terminus of the helical region, consistent with delayed folding of the molecule. Differential scanning calorimetry of patient collagens was consistent with an increased post-translational modification in the absence of a primary structural defect, with patient collagens showing a one degree centigrade increase in thermal stability compared to normal control collagen. Proband collagen secretion is moderately delayed but total collagen secretion is increased.[unreadable] [unreadable] For type VIII OI, we are investigating a special LEPRE1 mutation, IVS5+1G>T, which causes 5 alternatively spliced forms of the transcript, each leading to a PTC. Homozygotes for this mutation die within the first few months of life while compound heterozygotes survive with extremely severe bone disease. The mutation occurs only in African Americans and West Africans. We are interested in determining the incidence of carriers for this mutation and in understanding its molecular anthropology. We have used a restriction enzyme digestion assay and a custom SNP assay to identify carriers among contemporary African Americans from Pennsylvania, Maryland and DC, as well as contemporary West Africans. The carrier rate among these populations is 1/200-300 individuals, predicting an estimate incidence of type VIII OI from this mutation of 1/165000-320000. Among contemporary West Africans, we were surprised to find a carrier frequency of 1.37%. This very high incidence predicts that the rate of recessive OI in West Africa will be equal to the incidence of dominant OI. We are currently using haplotype analysis to determine the age of the mutation, which was presumably brought to the Americas via the Atlantic slave trade.[unreadable] [unreadable] These recessive null mutations of CRTAP and LEPRE1 result in a novel metabolic disorder of bone, and demonstrate that the 3-hydroxylation complex is crucial for normal bone development. Our work has generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in the components of a complex in the endoplasmic reticulum that modifies collagen cause recessive OI.